Acyl carrier protein promotes MukBEF action in Escherichia coli chromosome organization-segregation.

Structural Maintenance of Chromosomes (SMC) complexes act ubiquitously to compact DNA linearly, thereby facilitating chromosome organization-segregation. SMC proteins have a conserved architecture, with a dimerization hinge and an ATPase head domain separated by a long antiparallel intramolecular coiled-coil. Dimeric SMC proteins interact with essential accessory proteins, kleisins that bridge the two subunits of an SMC dimer, and HAWK/KITE proteins that interact with kleisins.

Competitive binding of MatP and topoisomerase IV to the MukB hinge domain.

Structural Maintenance of Chromosomes (SMC) complexes have ubiquitous roles in compacting DNA linearly, thereby promoting chromosome organization-segregation. Interaction between the SMC complex, MukBEF, and -bound MatP in the chromosome replication termination region, , results in depletion of MukBEF from , a process essential for efficient daughter chromosome individualization and for preferential association of MukBEF with the replication origin region. Chromosome-associated MukBEF complexes also interact with topoisomerase IV (ParCE), so that their chromosome distribution mirrors that of MukBEF.

Dynamic architecture of the Escherichia coli structural maintenance of chromosomes (SMC) complex, MukBEF.

Ubiquitous Structural Maintenance of Chromosomes (SMC) complexes use a proteinaceous ring-shaped architecture to organize and individualize chromosomes, thereby facilitating chromosome segregation. They utilize cycles of adenosine triphosphate (ATP) binding and hydrolysis to transport themselves rapidly with respect to DNA, a process requiring protein conformational changes and multiple DNA contact sites. By analysing changes in the architecture and stoichiometry of the Escherichia coli SMC complex, MukBEF, as a function of nucleotide binding to MukB and subsequent ATP hydrolysis, we demonstrate directly the formation of dimer of MukBEF dimer complexes, dependent on dimeric MukF kleisin.

MukB ATPases are regulated independently by the N- and C-terminal domains of MukF kleisin.

The SMC complex, MukBEF, acts in chromosome segregation. MukBEF shares the distinctive architecture of other SMC complexes, with one prominent difference; unlike other kleisins, MukF forms dimers through its N-terminal domain. We show that a 4-helix bundle adjacent to the MukF dimerisation domain interacts functionally with the MukB coiled-coiled 'neck' adjacent to the ATPase head.

Activation of Xer-recombination at dif: structural basis of the FtsKγ-XerD interaction.

Bacterial chromosomes are most often circular DNA molecules. This can produce a topological problem; a genetic crossover from homologous recombination results in dimerization of the chromosome. A chromosome dimer is lethal unless resolved.

Assembly, translocation, and activation of XerCD-dif recombination by FtsK translocase analyzed in real-time by FRET and two-color tethered fluorophore motion.

The FtsK dsDNA translocase functions in bacterial chromosome unlinking by activating XerCD-dif recombination in the replication terminus region. To analyze FtsK assembly and translocation, and the subsequent activation of XerCD-dif recombination, we extended the tethered fluorophore motion technique, using two spectrally distinct fluorophores to monitor two effective lengths along the same tethered DNA molecule. We observed that FtsK assembled stepwise on DNA into a single hexamer, and began translocation rapidly (∼ 0.

Single-molecule imaging of FtsK translocation reveals mechanistic features of protein-protein collisions on DNA.

In physiological settings, DNA translocases will encounter DNA-bound proteins, which must be dislodged or bypassed to allow continued translocation. FtsK is a bacterial translocase that promotes chromosome dimer resolution and decatenation by activating XerCD-dif recombination. To better understand how translocases act in crowded environments, we used single-molecule imaging to visualize FtsK in real time as it collided with other proteins.

Conformational transitions during FtsK translocase activation of individual XerCD-dif recombination complexes.

Three single-molecule techniques have been used simultaneously and in tandem to track the formation in vitro of single XerCD-dif recombination complexes. We observed the arrival of the FtsK translocase at individual preformed synaptic complexes and demonstrated the conformational change that occurs during their activation. We then followed the reaction intermediate transitions as Holliday junctions formed through catalysis by XerD, isomerized, and were converted by XerC to reaction products, which then dissociated.

Capturing reaction paths and intermediates in Cre-loxP recombination using single-molecule fluorescence.

Site-specific recombination plays key roles in microbe biology and is exploited extensively to manipulate the genomes of higher organisms. Cre is a well studied site-specific recombinase, responsible for establishment and maintenance of the P1 bacteriophage genome in bacteria. During recombination, Cre forms a synaptic complex between two 34-bp DNA sequences called loxP after which a pair of strand exchanges forms a Holliday junction (HJ) intermediate; HJ isomerization then allows a second pair of strand exchanges and thus formation of the final recombinant product.

The Escherichia coli DNA translocase FtsK.

Escherichia coli FtsK is a septum-located DNA translocase that co-ordinates the late stages of cytokinesis and chromosome segregation. Relatives of FtsK are present in most bacteria; in Bacillus subtilis, the FtsK orthologue, SpoIIIE, transfers the majority of a chromosome into the forespore during sporulation. DNA translocase activity is contained within a ~ 512-amino-acid C-terminal domain, which is divided into three subdomains: alpha, beta and gamma.

FtsK translocation on DNA stops at XerCD-dif.

Escherichia coli FtsK is a powerful, fast, double-stranded DNA translocase, which can strip proteins from DNA. FtsK acts in the late stages of chromosome segregation by facilitating sister chromosome unlinking at the division septum. KOPS-guided DNA translocation directs FtsK towards dif, located within the replication terminus region, ter, where FtsK activates XerCD site-specific recombination.

KOPS-guided DNA translocation by FtsK safeguards Escherichia coli chromosome segregation.

The septum-located DNA translocase, FtsK, acts to co-ordinate the late steps of Escherichia coli chromosome segregation with cell division. The FtsK gamma regulatory subdomain interacts with 8 bp KOPS DNA sequences, which are oriented from the replication origin to the terminus region (ter) in each arm of the chromosome. This interaction directs FtsK translocation towards ter where the final chromosome unlinking by decatenation and chromosome dimer resolution occurs.

The FtsK gamma domain directs oriented DNA translocation by interacting with KOPS.

The bacterial septum-located DNA translocase FtsK coordinates circular chromosome segregation with cell division. Rapid translocation of DNA by FtsK is directed by 8-base-pair DNA motifs (KOPS), so that newly replicated termini are brought together at the developing septum, thereby facilitating completion of chromosome segregation. Translocase functions reside in three domains, alpha, beta and gamma.

Dissection of a functional interaction between the DNA translocase, FtsK, and the XerD recombinase.

Successful bacterial circular chromosome segregation requires that any dimeric chromosomes, which arise by crossing over during homologous recombination, are converted to monomers. Resolution of dimers to monomers requires the action of the XerCD site-specific recombinase at dif in the chromosome replication terminus region. This reaction requires the DNA translocase, FtsK(C), which activates dimer resolution by catalysing an ATP hydrolysis-dependent switch in the catalytic state of the nucleoprotein recombination complex.

Functional analysis of the C-terminal domains of the site-specific recombinases XerC and XerD.

The tyrosine family site-specific recombinases XerC and XerD convert dimers of the Escherichia coli chromosome and many natural plasmids to monomers. The heterotetrameric recombination complex contains two molecules of XerC and two of XerD, with each recombinase mediating one pair of DNA strand exchanges. The two pairs of strand exchanges are separated in time and space.